Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA

We here report the isolation and nucleotide sequencing of a full-length 3.3-kilobase cDNA for the cytoplasmic form of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a regulated enzyme in the cholesterol biosynthetic pathway. The cDNA was isolated from UT-1 cells, a compactin-resistant line of Chinese hamster ovary cells. UT-1 cells produce large amounts of mRNA for HMG-CoA synthase and the next enzyme in the pathway, HMG-CoA reductase, as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. The identity of the cDNA for HMG-CoA synthase was confirmed through comparison of the NH2-terminal amino acid sequence predicted from the cDNA with that determined chemically from the purified enzyme. Anti-peptide antibodies directed against the amino acid sequence predicted from the cDNA precipitated HMG-CoA synthase activity from liver cytoplasm. The feeding of cholesterol to hamsters led to a decrease of more than 85% in the amount of mRNA for HMG-CoA synthase and HMG-CoA reductase in hamster liver. These data indicate that the mRNAs for cytoplasmic HMG-CoA synthase and for HMG-CoA reductase, two sequential enzymes in the cholesterol biosynthetic pathway, are coordinately regulated by cholesterol.     

Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells

32P-labeled cDNA probes were used to study levels of genomic DNA and regulation of mRNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT-1 cells, a clone of compactin-resistant Chinese hamster ovary cells that have a 100-1000-fold increase in the amount of reductase protein. Similar measurements were made for the 53-kDa protein, a cytosolic protein of unknown function that is also expressed at high levels in UT-1 cells. The number of copies of the gene for reductase was increased by 15-fold in UT-1 cells as compared to the parental Chinese hamster ovary cells, as judged from Southern gel analysis of restriction endonuclease-digested genomic DNA. In contrast, there was no detectable increase in the number of gene copies for the 53-kDa protein. The amount of cytoplasmic mRNA for both proteins was markedly elevated in UT-1 cells, as determined by filter hybridization studies using 32P-labeled cDNA probes. The amount of mRNA for both reductase and the 53-kDa protein declined in parallel after addition of low density lipoprotein, 25-hydroxycholesterol, or mevalonate to the culture medium. The decline in reductase mRNA was associated with a marked decrease in the rate of [3H]uridine incorporation into hybridizable cytoplasmic mRNA. When UT-1 cells were grown for 3-4 months in the absence of compactin, the level of reductase mRNA and enzymatic activity decreased markedly, but the number of copies of the reductase gene did not decline. When the compactin-withdrawn cells were rechallenged with compactin, high levels of reductase mRNA and enzymatic activity promptly returned. We conclude that the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, has been stably amplified in UT-1 cells. Despite this differential gene amplification, the levels of cytoplasmic mRNA for both gene products are markedly elevated, and both are reduced in parallel by either sterols (low density lipoprotein-cholesterol or 25-hydroxycholesterol) or mevalonate, the product of the reductase-catalyzed reaction.     

Effects of compactin on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in compactin-resistant C100 and wild-type cells

A cell line, C100, resistant to 225 μm compactin, has been isolated which overproduces 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase approximately 100-fold compared to the parental cell line [E. Hardeman, H. Jenke and R. Simoni (1983) Proc. Natl. Acad. Sci. U.S.A.80, 1516–1520]. It is demonstrated that the overproduction of HMG-CoA reductase in these cells is the result of increased enzyme synthesis due to elevated levels of translatable mRNA. Furthermore, the apparent molecular weight of the in vitro translation product is 94,000, which agrees with the molecular weight of the in vivo synthesized HMG-CoA reductase protomer in C100 cells. However, a comparison of the Staphylococcus aureus V8 proteolysis patterns between the in vitro and in vivo translation products reveals structural differences which suggests in vivo posttranslation modification(s). It is also demonstrated unequivocally, by comparing proteolytic cleavage patterns and pulse-chase experiments, that the previously reported 63,000-, 52,000-, and 38,000-Da polypeptides recognized by HMG-CoA reductase antiserum derive from the 94,000-Da protomer as a result of nonphysiological proteolysis. Finally, the types of regulatory mechanisms involved in both the induction and repression of the enzyme in the presence or absence of compactin were determined. Four biochemical parameters of HMG-CoA reductase were examined in variant and parental cells grown in the presence and absence of compactin: enzymatic activity, degradation rate, synthesis rate, and concentration of translatable mRNA. These studies revealed that changes in cellular HMG-CoA reductase content are a function of concurrent changes in the rates of enzyme degradation and synthesis. Changes in enzyme synthesis are due to alterations in the level of translatable mRNA.     

Effects of compactin, mevalonate and low-density lipoprotein on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2.

Compactin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, decreased cholesterol synthesis in intact Hep G2 cells. However, after the inhibitor was washed away, the HMG-CoA-reductase activity determined in the cell homogenate was found to be increased. Also the high-affinity association of LDL (low-density lipoprotein) to Hep G2 cells was elevated after incubation with compactin. Lipoprotein-depleted serum, present in the incubation medium, potentiated the compactin effect compared with incubation in the presence of human serum albumin. Addition of either mevalonate or LDL prevented the compactin-induced rise in activities of both HMG-CoA reductase and LDL receptor in a comparable manner. It is concluded that in this human hepatoma cell line, as in non-transformed cells, both endogenous mevalonate or mevalonate-derived products and exogenous cholesterol are able to modulate the HMG-CoA reductase activity as well as the LDL-receptor activity.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant Chinese hamster ovary cell line

Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (CR-7), and revertant (RCR-7) cells. In contrast to WT and RCR-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of R-[2-14C]mevalonolactone revealed that CR-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but CR-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of 125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of 125I-LDL were also similar, suggesting that CR-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1-2 micrograms/ml) strongly and reversibly suppressed reductase activity in WT and RCR-7. CR-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of 3H-labeled mannose incorporation into cellular protein, but 3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.     

Altered function and structure of low-density lipoprotein receptor in compactin (ML236B)-resistant mutants of Chinese hamster cells

Mutants resistant to compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, have been previously isolated from the Chinese hamster V79 cell line. Two compactin-resistant mutants, MF-1 and MF-2, show altered responses to human low-density lipoprotein (LDL). Accumulation of fluorescent-labeled LDL was much reduced. Ligand blotting showed LDL receptor activity in MF-1 and MF-2 cells of about one half to one third that of V79. Internalization and degradation of LDL in MF-1 or MF-2 cells were about one tenth those in V79 cells, suggesting that the LDL binding as well as the LDL internalization of the compactin-resistant clones was altered. Down-regulation of LDL receptor activity as well as hydroxymethylglutaryl CoA reductase was observed in V79 cells treated with LDL, while there appeared to be much less down-regulation in MF-1 and MF-2 cells. Using anti-LDL receptor antibody, MF-1 and MF-2 cells were found to produce smaller-sized mature forms of LDL receptor: the molecular mass of the mutant LDL receptor was 3-5 kDa smaller than that of the parental LDL receptor. Altered O-linked oligosaccharides or amino acid sequence might account for the decreased molecular mass and aberrant properties of the LDL receptor in MF-1 and MF-2.     

Compactin and simvastatin, but not pravastatin, induce bone morphogenetic protein-2 in human osteosarcoma cells

Bone morphogenetic protein (BMP)-2, a member of the BMP family, plays an important role in osteoblast differentiation and bone formation. To discover small molecules that induce BMP-2, a luciferase reporter vector containing the 5'-flanking promoter region of the human BMP-2 gene was constructed and transfected into human osteosarcoma (HOS) cells. By the screening of an in-house natural product library with stably transfected HOS cells, a fungal metabolite, compactin, known as an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was isolated. The stimulation of the promoter activity by compactin seemed to be specific for BMP-2 gene in HOS cells, since it had little effect on BMP-4 or SV40 promoter activity and the stimulation was not observed in Chinese hamster ovary (CHO) cells. RT-PCR analysis and alkaline phosphatase assay revealed that compactin induced an increase in the expression of BMP-2 mRNA and protein. Like compactin, simvastatin also activated the BMP-2 promoter, whereas pravastatin did not. The statin-mediated activation of BMP-2 promoter was completely inhibited by the downstream metabolite of HMG-CoA reductase, mevalonate, indicating that the activation was a result of the inhibition of the enzyme. These results suggest that statins, if they are selectively targeted to bone, have beneficial effects in the treatment of osteoporosis or bone fracture.     

Regulation of cholesterol synthesis in cultured mouse mammary carcinoma FM3A cells

Mouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 micrograms protein/ml) resulted in a 50% decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium. ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 microM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 microM for 24 h). Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.     

Somatic cell genetics and the study of cholesterol metabolism

The regulation of cholesterol biosynthesis by extracellular cholesterol occurs both in whole animal tissue and in permanent somatic cell lines in culture. Permanent mammalian cells lines, under optimized growth conditions, are easily manipulated both biochemically and genetically. The Chinese hamster ovary cell line (CHO-K1) is the most widely used cell line for genetic studies. CHO-K1 is a pseudo-diploid mammalian cell exhibiting a short doubling time and a relatively high plating efficiency. Somatic cell mutants can be generated through mutagenesis and also by drug adaptation. Following mutagenesis, auxotrophs may be isolated either by selection or by screening. Most selection procedures for mutants of cholesterol metabolism must be done in serum depleted of cholesterol which requires the endogenous biosynthetic pathway to be intact. Mutants failing to produce cholesterol do not replicate their DNA and exhibit reduced concentrations of cholesterol in their membranes. BUdR and polyene antibiotics have both been used to select against the wild-type cells which incorporate these compounds and are killed, allowing the survival of the mutant cells. Both mevalonate and cholesterol auxotrophs have been isolated with the BUdR technique and have proven useful for elucidation of the early steps in cholesterol biosynthesis, particularly for the ratelimiting enzyme HMG-CoA reductase. Somatic cell fusion of a mutant and wild-type cell followed by chromosomal segregation, routinely used to map human genes, has also been used to map the human gene for HMG-CoA synthase. Such hybrids also provide valuable information on the dominance or recessivity of a specific lesion. DNA-mediated gene transfer into somatic cell mutants allows the selection of DNA sequences which complement the mutation, and is also useful for analysis of regions of regulatory significance. Mutants, resistant to the regulatory effects of oxygenated sterols, can be isolated following mutagenesis. Mutants of this type vary the lipid content of their membranes in response to cholesterol concentration in the medium. All such mutants tested exhibit a pleiotropic regulatory effect on more than one enzyme in the cholesterol biosynthetic pathway. Adaptation to drugs such as compactin and mevinolin, which inhibit HMG-CoA reductase, have been used to produce mutants which overexpress enzymes in the pathway. These amplified cells are useful sources of specific mRNAs for construction of cDNA libraries and gene isolation. Structure-function relationships of membrane sterols can be studied in cholesterol auxotrophs where changes in acyl-chain ordering can be manipulated by exogenous sterols in the medium.     

Farnesyl Acetate, a Derivative of an Isoprenoid of the Mevalonate Pathway, Inhibits DNA Replication in Hamster and Human Cells

Cells treated with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme which catalyzes the rate-limiting step of the mevalonate pathway, are arrested prior to the DNA synthesis (S) phase of the cell cycle. Identification of a specific pathway product or products with a role in DNA replication, however, has remained elusive. In this report we demonstrate that farnesyl acetate, a derivative of the key isoprenoid pathway intermediate farnesyl pyrophosphate, inhibits DNA replication in both Chinese hamster ovary cells and human (HeLa) cells. This effect is revealed by measurement of DNA content using fluorescence-activated cell sorter analysis and by measurement of [³H]thymidine incorporation. We show that cells treated with farnesyl acetate retain protein synthesis capacity as DNA replication is inhibited and remain intact as viewed with the vital stain propidium iodide. The inhibition of DNA replication by farnesyl acetate occurs in cells treated with high levels of compactin and in cells lacking HMG-CoA reductase. These results indicate that farnesyl acetate action is not dependent on metabolism through the isoprenoid pathway and is not the result of the loss of a metabolite required for replication nor the accumulation of a metabolite which is inhibitory. In addition, cells treated with farnesyl acetate for over 6 h are irreversibly blocked from progressing through S phase, a phenomenon which differs sharply from the results with compactin, removal of which results in synchronous progression through S phase. Farnesyl acetate also blocks protein prenylation in cells, to a degree comparable to a known farnesylation inhibitor, BZA-5B. We propose that farnesyl acetate is acting in a manner quite different from the metabolic block caused by compactin, causing a rapid and irreversible block of DNA replication.     

Squalene synthase-deficient mutant of Chinese hamster ovary cells.

Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation.     

Independent regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and chylomicron remnant receptor activities in rat liver.

Treatment of rats with pharmacological doses of oestrogen resulted in a 3-fold decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and a 4-fold increase in saturable binding of 125I-labelled chylomicron remnants to liver membranes in vitro. Intragastric administration of mevalonolactone to rats did not affect the capacity of the liver membranes to bind to labelled chylomicron remnants even though there was a substantial decrease in the activity of HMG-CoA reductase. Similar results were obtained after cholesterol feeding. Simultaneous treatment of rats with cholestyramine and compactin increased hepatic HMG-CoA reductase activity 6-fold. However, liver membranes derived from these animals showed no change in their capacity to bind to labelled chylomicron remnants in vitro. Administration of mevalonolactone to the cholestyramine/compactin-treated animals also failed to produce a change in remnant-binding capacity. Although administration of mevalonolactone alone produced a significant 3-fold decrease in the activity of hepatic HMG-CoA reductase it was unable to suppress significantly the increase in enzyme activity caused by treatment with cholestyramine and compactin.